Probiotic bifidobacterium strain

ABSTRACT

Probiotic  Bifidobacterium  strain AH1714 is significantly immunomodulatory following oral consumption. The strain is useful as an immunomodulatory biotherapeutic agent.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of prior InternationalApplication No. PCT/IE2010/000066 filed on Nov. 11, 2010 (aka 12424& WO)which claims the benefit of U.S. application Ser. No. 12/616,752 (aka11484M) filed Nov. 11, 2009 and U.S. Provisional Application No.61/344,030 (aka 12424P&) filed May 11, 2010.

INTRODUCTION

The invention relates to a Bifidobacterium strain and its use as aprobiotic bacteria in particular as an immunomodulatory biotherapeuticagent.

The defense mechanisms to protect the human gastrointestinal tract fromcolonization by intestinal bacteria are highly complex and involve bothimmunological and non-immunological aspects (1). Innate defensemechanisms include the low pH of the stomach, bile salts, peristalsis,mucin layers and anti-microbial compounds such as lysozyme (2).Immunological mechanisms include specialized lymphoid aggregates,underlying M cells, called peyers patches which are distributedthroughout the small intestine and colon (3). Luminal antigens presentedat these sites result in stimulation of appropriate T and B cell subsetswith establishment of cytokine networks and secretion of antibodies intothe gastrointestinal tract (4). In addition, antigen presentation mayoccur via epithelial cells to intraepithelial lymphocytes and to theunderlying lamina propria immune cells (5). Therefore, the host investssubstantially in immunological defense of the gastrointestinal tract.However, as the gastrointestinal mucosa is the largest surface at whichthe host interacts with the external environment, specific controlmechanisms must be in place to regulate immune responsiveness to the 100tons of food which is handled by the gastrointestinal tract over anaverage lifetime. Furthermore, the gut is colonized by over 500 speciesof bacteria numbering 10¹¹-10¹²/g in the colon. Thus, these controlmechanisms must be capable of distinguishing non-pathogenic adherentbacteria from invasive pathogens, which would cause significant damageto the host. In fact, the intestinal flora contributes to defense of thehost by competing with newly ingested potentially pathogenicmicro-organisms.

Bacteria present in the human gastrointestinal tract can promoteinflammation. Aberrant immune responses to the indigenous microflorahave been implicated in certain disease states, such as inflammatorybowel disease. Antigens associated with the normal flora usually lead toimmunological tolerance and failure to achieve this tolerance is a majormechanism of mucosal inflammation (6). Evidence for this breakdown intolerance includes an increase in antibody levels directed against thegut flora in patients with inflammatory bowel disease (IBD).

The present invention is directed towards a Bifidobacterium strain whichhas been shown to have immunomodulatory effects, by modulating cytokinelevels or by antagonizing and excluding pro-inflammatory micro-organismsfrom the gastrointestinal tract.

STATEMENTS OF INVENTION

The invention provides an isolated strain of Bifidobacterium NCIMB41676.

The Bifidobacterium strain may be in the form of viable cells. TheBifidobacterium strain may be in the form of non-viable cells. TheBifidobacterium may be isolated from colonic biopsy tissue from ahealthy human subject. The Bifidobacterium strain may be significantlyimmunomodulatory following oral consumption in humans.

The invention also provides a formulation which comprises aBifidobacterium strain as described herein. The formulation may furthercomprise a probiotic material. The formulation may further comprise aprebiotic material. The formulation may further comprise an ingestablecarrier. The ingestable carrier may a pharmaceutically acceptablecarrier such as a capsule, tablet or powder. The ingestable carrier maybe a food product such as acidified milk, yoghurt, frozen yoghurt, milkpowder, milk concentrate, cheese spreads, dressings or beverages. Theformulation may further comprise a protein and/or peptide, in particularproteins and/or peptides that are rich in glutamine/glutamate, a lipid,a carbohydrate, a vitamin, mineral and/or trace element. TheBifidobacterium strain may be present in an amount of more than 10⁶ cfuper gram of the formulation. The formulation may further comprise anadjuvant. The formulation may further comprise a bacterial component.The formulation may further comprise a drug entity. The formulation mayfurther comprise a biological compound. The formulation may be used forimmunisation and vaccination protocols.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in foodstuffs.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use as a medicament.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment ofundesirable inflammatory activity.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment ofundesirable gastrointestinal inflammatory activity such as inflammatorybowel disease eg. Crohns disease or ulcerative colitis, irritable bowelsyndrome; pouchitis; or post infection colitis.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment ofgastrointestinal cancer(s).

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment of systemicdisease such as rheumatoid arthritis.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment ofautoimmune disorders due to undesirable inflammatory activity.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment of cancerdue to undesirable inflammatory activity.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis of cancer.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment ofdiarrhoeal disease due to undesirable inflammatory activity, such asClostridium difficile associated diarrhoea, Rotavirus associateddiarrhoea or post infective diarrhoea or diarrhoeal disease due to aninfectious agent, such as E. coli.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the preparation of anti-inflammatorybiotherapeutic agents for the prophylaxis and/or treatment ofundesirable inflammatory activity.

Bifidobacterium strains as described herein may be used in thepreparation of a panel of biotherapeutic agents for modifying the levelsof IL-10.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prevention and/or treatment ofinflammatory disorders, immunodeficiency, inflammatory bowel disease,irritable bowel syndrome, cancer (particularly of the gastrointestinaland immune systems), diarrhoeal disease, antibiotic associateddiarrhoea, paediatric diarrhoea, appendicitis, autoimmune disorders,multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, coeliacdisease, diabetes mellitus, organ transplantation, bacterial infections,viral infections, fungal infections, periodontal disease, urogenitaldisease, sexually transmitted disease, HIV infection, HIV replication,HIV associated diarrhoea, surgical associated trauma, surgical-inducedmetastatic disease, sepsis, weight loss, anorexia, fever control,cachexia, wound healing, ulcers, gut barrier function, allergy, asthma,respiratory disorders, circulatory disorders, coronary heart disease,anaemia, disorders of the blood coagulation system, renal disease,disorders of the central nervous system, hepatic disease, ischaemia,nutritional disorders, osteoporosis, endocrine disorders, epidermaldisorders, psoriasis, acne vulgaris, panic disorder, behavioral disorderand/or post traumatic stress disorders.

The Bifidobacterium strain as described herein may act by antagonisingand excluding proinflammatory micro-organisms from the gastrointestinaltract.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the preparation of anti-inflammatorybiotherapeutic agents for reducing the levels of pro inflammatorycytokines.

The Bifidobacterium strain as described herein may be used as ananti-infective probiotic strain.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment of bipolarillness, depression, mood disorders, and/or anxiety disorders.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein may be used as a cognative enhancer for the prophylaxisand/or treatment of disorders of the central nervous system such asAlzheimer's disease, schizophrenia and/or mild cognative disorder.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment of obesityrelated inflammation.

The invention also provides a Bifidobacterium strain or a formulation asdescribed herein for use in the prophylaxis and/or treatment of obesityrelated metabolic dysregulation.

We describe Bifidobacterium strain AH1714 (NCIMB 41676) or mutants orvariants thereof. The mutant may be a genetically modified mutant. Thevariant may be a naturally occurring variant of Bifidobacterium. Alsodescribed is a rifampicin resistant variant of strain AH1714. The strainmay be a probiotic. It may be in the form of a biologically pureculture.

We also describe an isolated strain of Bifidobacterium NCIMB 41676. TheBifidobacterium strains may be in the form of viable cells.Alternatively Bifidobacterium strains are in the form of non-viablecells. The general use of probiotic bacteria is in the form of viablecells. However, it can also be extended to non-viable cells such askilled cultures or compositions containing beneficial factors expressedby the probiotic bacteria. This could include thermally killedmicro-organisms or micro-organisms killed by exposure to altered pH orsubjection to pressure or gamma irradiation. With non-viable cellsproduct preparation is simpler, cells may be incorporated easily intopharmaceuticals and storage requirements are much less limited thanviable cells. Lactobacillus casei YIT 9018 offers an example of theeffective use of heat killed cells as a method for the treatment and/orprevention of tumour growth as described in U.S. Pat. No. 4,347,240.

The Bifidobacterium strains may be isolated from colonic biopsy tissuefrom healthy human subjects, the Bifidobacterium strains beingsignificantly immunomodulatory following oral consumption in humans.

We also describe a formulation which comprises the Bifidobacteriumstrain as described herein. The formulation may include anotherprobiotic material. The formulation may include a prebiotic material.Preferably the formulation includes an ingestable carrier. Theingestable carrier may be a pharmaceutically acceptable carrier such asa capsule, tablet or powder. Preferably the ingestable carrier is a foodproduct such as acidified milk, yoghurt, frozen yoghurt, milk powder,milk concentrate, cheese spreads, dressings or beverages. Theformulation may further comprise a protein and/or peptide, in particularproteins and/or peptides that are rich in glutamine/glutamate, a lipid,a carbohydrate, a vitamin, mineral and/or trace element. TheBifidobacterium strain may be present in the formulation at more than10⁶ cfu per gram of delivery system. Preferably the formulation includesany one or more of an adjuvant, a bacterial component, a drug entity ora biological compound.

We also describe a Bifidobacterium strain or a formulation for use asfoodstuffs, as a medicament, for use in the prophylaxis and/or treatmentof undesirable inflammatory activity, for use in the prophylaxis and/ortreatment of undesirable respiratory inflammatory activity such asasthma, for use in the prophylaxis and/or treatment of undesirablegastrointestinal inflammatory activity such as inflammatory boweldisease eg. Crohns disease or ulcerative colitis, irritable bowelsyndrome, pouchitis, or post infection colitis, for use in theprophylaxis and/or treatment of gastrointestinal cancer(s), for use inthe prophylaxis and/or treatment of systemic disease such as rheumatoidarthritis, for use in the prophylaxis and/or treatment of autoimmunedisorders due to undesirable inflammatory activity, for use in theprophylaxis and/or treatment of cancer due to undesirable inflammatoryactivity, for use in the prophylaxis of cancer, for use in theprophylaxis and/or treatment of diarrhoeal disease due to undesirableinflammatory activity, such as Clostridium difficile associateddiarrhoea, Rotavirus associated diarrhoea or post infective diarrhoea,for use in the prophylaxis and/or treatment of diarrhoeal disease due toan infectious agent, such as E. coli.

We also describe a Bifidobacterium strain or a formulation of theinvention for use in the preparation of an anti-inflammatorybiotherapeutic agent for the prophylaxis and/or treatment of undesirableinflammatory activity or for use in the preparation of anti-inflammatorybiotherapeutic agents for the prophylaxis and/or treatment ofundesirable inflammatory activity. The strain may act by antagonisingand excluding proinflammatory micro-organisms from the gastrointestinaltract.

We also describe a Bifidobacterium strain or a formulation for use inthe preparation of anti-inflammatory biotherapeutic agents for reducingthe levels of pro-inflammatory cytokines.

The Bifidobacterium strain may be used in the preparation ofanti-inflammatory biotherapeutic agents for modifying the levels ofIL-10.

The Bifidobacterium strain may be used as a anti-infective probiotic dueto their ability to antagonise the growth of pathogenic species.

We have found that particular strains of Bifidobacterium elicitimmunomodulatory effects in vitro.

The invention is therefore of major potential therapeutic value in theprophylaxis or treatment of dysregulated immune responses, such asundesirable inflammatory reactions for example asthma.

Bifidobacterium are commensal microorganisms. They have been isolatedfrom the microbial flora within the human gastrointestinal tract. Theimmune system within the gastrointestinal tract cannot have a pronouncedreaction to members of this flora, as the resulting inflammatoryactivity would also destroy host cells and tissue function. Therefore,some mechanism(s) exist whereby the immune system can recognizecommensal non-pathogenic members of the gastrointestinal flora as beingdifferent to pathogenic organisms. This ensures that damage to hosttissues is restricted and a defensive barrier is still maintained.

A deposit of Bifidobacterium longum strain AH1714 was made at theNational Collections of Industrial and Marine Bacteria Limited (NCIMB)Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,Scotland, UK on Nov. 5, 2009 and accorded the accession number NCIMB41676.

The Bifidobacterium longum may be a genetically modified mutant or itmay be a naturally occurring variant thereof.

Preferably the Bifidobacterium longum may be in the form of viablecells.

Alternatively the Bifidobacterium longum may be in the form ofnon-viable cells.

It will be appreciated that the specific Bifidobacterium strain of theinvention may be administered to animals (including humans) in an orallyingestible form in a conventional preparation such as capsules,microcapsules, tablets, granules, powder, troches, pills, suppositories,suspensions and syrups. Suitable formulations may be prepared by methodscommonly employed using conventional organic and inorganic additives.The amount of active ingredient in the medical composition may be at alevel that will exercise the desired therapeutic effect.

The formulation may also include a bacterial component, a drug entity ora biological compound.

In addition a vaccine comprising the strains of the invention may beprepared using any suitable known method and may include apharmaceutically acceptable carrier or adjuvant.

Throughout the specification the terms mutant, variant and geneticallymodified mutant include a strain of Bifidobacteria whose genetic and/orphenotypic properties are altered compared to the parent strain.Naturally occurring variant of Bifidobacterium longum includes thespontaneous alterations of targeted properties selectively isolated.Deliberate alteration of parent strain properties is accomplished byconventional (in vitro) genetic manipulation technologies, such as genedisruption, conjugative transfer, etc. Genetic modification includesintroduction of exogenous and/or endogenous DNA sequences into thegenome of a Bifidobacteria strain, for example by insertion into thegenome of the bacterial strain by vectors, including plasmid DNA, orbacteriophages.

Natural or induced mutations include at least single base alterationssuch as deletion, insertion, transversion or other DNA modificationswhich may result in alteration of the amino acid sequence encoded by theDNA sequence.

The terms mutant, variant and genetically modified mutant also include astrain of Bifidobacteria that has undergone genetic alterations thataccumulate in a genome at a rate which is consistent in nature for allmicro-organisms and/or genetic alterations which occur throughspontaneous mutation and/or acquisition of genes and/or loss of geneswhich is not achieved by deliberate (in vitro) manipulation of thegenome but is achieved through the natural selection of variants and/ormutants that provide a selective advantage to support the survival ofthe bacterium when exposed to environmental pressures such asantibiotics. A mutant can be created by the deliberate (in vitro)insertion of specific genes into the genome which do not fundamentallyalter the biochemical functionality of the organism but whose productscan be used for identification or selection of the bacterium, forexample antibiotic resistance.

A person skilled in the art would appreciate that mutant or variantstrains of Bifidobacteria can be identified by DNA sequence homologyanalysis with the parent strain. Strains of Bifidobacteria having aclose sequence identity with the parent strain are considered to bemutant or variant strains. A Bifidobacteria strain with a sequenceidentity (homology) of 96% or more, such as 97% or more, or 98% or more,or 99% or more with the parent DNA sequence may be considered to be amutant or variant. Sequence homology may be determined using on-linehomology algorithm “BLAST” program, publicly available athttp://www.ncbi.nlm.nih,gov/BLAST/.

Mutants of the parent strain also include derived Bifidobacteria strainshaving at least 85% sequence homology, such as at least 90% sequencehomology, or at least 95% sequence homology to the 16s-23s intergenicspacer polynucleotide sequence of the parent strain. These mutants mayfurther comprise DNA mutations in other DNA sequences in the bacterialgenome.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more clearly understood from the followingdescription thereof given by way of example only with reference to theaccompanying drawings in which;—

FIG. 1 is a graph illustrating transit of B. longum AH1714 through thegastrointestinal tract;

FIG. 2 is a photograph of B. longum AH1714 grown on a Congo Red Agarplate;

FIG. 3 is a bar chart illustrating the IL-10:IL-12p70 ratio for PBMCsstimulated with Bifidobacterium longum strain 1714 (Bifidobacterium1714);

FIG. 4 is a bar chart showing the induction profile of IL-10 insplenocytes isolated from both 1714 and PBS fed mice with and without invivo LPS challenge 1 mg/kg. In vitro cells are either unstimulated (A),stimulated with LPS (B) or stimulated with antiCD3/CD28 (C). Data isshown as Average & SEM;

FIG. 5 is a bar chart showing the induction profile of TNF-α insplenocytes isolated from both 1714 and PBS fed mice with and without invivo LPS challenge 1 mg/kg. In vitro cells are either unstimulated (A)or stimulated with antiCD3/CD28 (B). Data is shown as Average & SEM;

FIG. 6 is a bar chart showing the induction profile of IFN-γ insplenocytes isolated from both 1714 and PBS fed mice with and without invivo LPS challenge 1 mg/kg. In vitro cells are either unstimulated (A)or stimulated with antiCD3/CD28 (B). Data is shown as Average & SEM.

FIG. 7 is a bar chart showing the induction profile of IL-12p70 insplenocytes isolated from both 1714 and PBS fed mice with and without invivo LPS challenge 1 mg/kg. In vitro cells are either unstimulated (A)or stimulated with antiCD3/CD28 (B). Data is shown as Average & SEM;

FIG. 8 is a bar chart showing the induction profile of TNF-α (A) andIL-10 (B) in serum sampled from both 1714 and PBS fed mice post 2H invivo challenge with LPS 1 mg/kg. Data is shown as Average & SEM;

FIG. 9 is a bar chart showing NFkB activity (Photons/second) fromisolated spleen 3 hours post challenge with a single 0.5 mg/kg dose ofLPS, from Placebo and 1714-fed animals (** designates p<0.01);

FIG. 10 is a bar chart (A) showing NFkB activity (Photons/second) fromwhole body imaging 1.5 hours post challenge with a single 0.5 mg/kg doseof LPS, from Placebo and 1714-fed animals ((B) and (C) are whole bodyrepresentative images in black and white and colour;

FIG. 11 is a bar chart representing the time of immobility displayed bythe mice over a 6-min test;

FIG. 12 is a line graph representing the freezing percentage in responseto the fearful stimulus (context) for day 1 (acquisition), day 2(memory/extinction) and day 3 (extinction);

FIG. 13 is a line graph representing the freezing percentage in responseto the fearful stimulus (cue) for day 1 (acquisition), day 2(memory/extinction) and day 3 (extinction);

FIG. 14 is a bar chart representing the number of marbles buried by themice over a 30-min session;

FIG. 15 is a bar chart representing the body temperature variation (ΔT)mice displayed following handling; and

FIG. 16 is a bar chart showing the changes in cytokine levels instimulated splenocytes from the Diet Induced Mouse model.

DETAILED DESCRIPTION OF THE INVENTION

A deposit of Bifidobacterium longum strain AH1714 was made at theNational Collections of Industrial and Marine Bacteria Limited (NCIMB)Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,Scotland, UK on Nov. 5, 2009 and accorded the accession number NCIMB41676.

A deposit of Bifidobacterium longum strain UCC35624 was made at theNational Collections of Industrial and Marine Bacteria Limited (NCIMB)Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,Scotland, UK on Jan. 13, 1999 and accorded the accession number NCIMB41003.

EXAMPLES

The following examples further describe and demonstrate embodimentswithin the scope of the invention. The examples are given solely for thepurpose of illustration and are not to be construed as limitations ofthe present invention, as many variations thereof are possible withoutdeparting from the spirit and scope of the invention.

Example 1 Isolation of Bifidobacterium longum AH1714

Bifidobacterium longum strain AH1714 was isolated from colonic biopsytissue from healthy human subjects.

Sections of the large of the human G.I.T, obtained during colorectalscoping, were screened for probiotic bacterial strains. Musocal tissuefrom the human gastrointestinal tract was transferred to a collectiontube containing Phosphate Buffered Saline (PBS), supplemented with 0.05%cysteine-HCl). Triton X-100 (0.05%) was added to release the adherentmicroorganisms from the tissue sample. Tissue samples were thenincubated for 10 min. The samples were vortexed vigorously and adherentLactobacilli and Bifidobacteria isolated from the gastrointestinaltissue by plating on selective agar (De Man, Rogosa and Sharpe (MRS)agar+Vancomycin and Wilkins-Chalgren Agar+Mupirocin, respectively).Isolated colonies were picked from the plates and re-streaked threetimes to ensure purity. Microscope examination, Gram staining, Catalasetesting, Fructose-6-Phosphate Phosphoketolase assessment were used todetermine presumptive Bifidobacteria species and isolates were stockedin 40% glycerol and stored at −20° and −80° C. 16S intergenic spacerregion sequencing were used to confirm the identity of the newlyisolated strains.

Following isolation of a pure bifidobacteria strain, assigned thedesignation AH1714, microbiological characteristics were assessed andare summarized in Table 1 below. AH1714 is a gram positive, catalasenegative pleomorphic shaped bacterium which is Fructose-6-PhoshatePhosphoketolase positive, confirming its identity as a bifidobacterium.

TABLE 1 Physiochemical characteristics of B. longum AH1714 StrainCharacteristics B. longum AH1714 Gram Stain + Catalase − Motility −F6PPK* +16s-23s intergenic spacer (IGS) sequencing was performed to identify thespecies of Bifidobacteria isolated. Briefly, DNA was isolated fromAH1714 using 100 μl of Extraction Solution and 25 μl of TissuePreparation solution (Sigma, XNAT2 Kit). The samples were incubated for5 minutes at room temperature followed by 2 hrs at 95° C. and then 100μl of Neutralization Solution (Sigma, XNAT2 kit) was added. Genomic DNAsolution was quantified using a Nanodrop spectrophotometer and stored at4° C. PCR was performed using the IGS primers. The primer pairs usedwere IGS R 5′-CTGGTGCCAAGGCATCCA-3′ (SEQ ID No. 4) and IGS L5′-GCTGGATCACCTCCTTTCT-3′ (SEQ ID No. 3). The cycling conditions were94° C. for 4 min (1 cycle), 94° C. for 45 sec, 53° C. for 45 sec, 72° C.for 45 sec (28 cycles). The PCR reaction contained 2 μl (100 ng) of DNA,PCR mix (Sigma, Red Taq), 0.025 nM IGS L and R primer (MWG Biotech,Germany). The PCR reactions were performed on a Biotherma thermocycler.The PCR products (10 μl) were ran alongside a molecular weight marker(100 bp Ladder, Roche) on a 2% agarose EtBr stained gel in TAE, todetermine the IGS profile. PCR products of Bifidobacterium (single band)were purified using the Promega Wizard PCR purification kit. Thepurified PCR products were sequenced using the primer sequences (above)for the intergenic spacer region. Sequence data was then searchedagainst the NCBI nucleotide database to determine the identity of thestrain by nucleotide homology. The resultant DNA sequence data wassubjected to the NCBI standard nucleotide-to-nucleotide homology BLASTsearch engine (http://www.ncbi.nlm.nih.gov/BLAST/). The nearest match tothe sequence was identified and then the sequences were aligned forcomparison using DNASTAR MegAlign software. The sequences (SEQ ID NO. 1[IGS forward sequence] and SEQ ID NO. 2 [IGS reverse sequence]) obtainedcan be viewed in the sequence listing. Searching the NCIMB databaserevealed that AH1714 has a unique IGS (SEQ ID NO. 1 [forward sequence]and SEQ ID NO. 2 [reverse sequence]) sequence with its closest sequencehomology to a Bifidobacterium longum.

In order to develop a barcode PCR profile for AH1714, PCR was performedusing BOX primers (8). The cycling conditions were 94° C. for 7 min (1cycle); 94° C. for 1 minute, 53° C. for 45 secs, 65° C. for 8 minutes,(30 cycles) and 65° C. for 16 minutes. The PCR reaction contained 50 ngof DNA, PCR mix (Sigma, Red Taq) and 0.03 nM BOXA1R primer(5′-CTACGGCAAGGCGACGCTGACG-3′) (SEQ ID NO. 5) (MWG Biotech, Germany).The PCR reactions were performed on a Biotherma thermocycler. The PCRproducts were run on a 3% agarose gel alongside a molecular weightmarker (Roche, 100 bp ladder) and imaged.

Antibiotic Sensitivity Profiles

The antibiotic sensitivity profiles for B. longum AH1714 was determinedusing the ‘disc susceptibility’ assay. The cultures were grown up in theappropriate broth medium for 48 h and spread-plated (100 μl) onto agarmedia. Discs containing known concentrations of the antibiotics wereplaced onto the agar. Strains were examined for antibiotic sensitivityafter 1-2 days incubation at 37° C. under anaerobic conditions.

TABLE 2 antibiotic resistance Antibiotic Group AH1714 Penicillin Gβ-lactam antibiotic S Ampicillin β-lactam antibiotic S Methicillinβ-lactam antibiotic M Streptomycin Aminoglycoside antibiotic RGentamicin Aminoglycoside antibiotic M Vancomycin Glycopeptideantibiotic S Nalidixic Acid Synthetic quinolone antibiotic R NovobiocinAminocoumarin antibiotic S Tetracycline Polyketide antibiotic SSulphamethoxazole Sulfonamide antibiotic R Trimethoprim\ Sulfonamideantibiotic R Sulphamethoxazole Trimethoprim R Rifampicin Rifamycinantibiotic S Chloramphenicol S Metronidazole Nitroimidazole antibioticsM Mupirocin R R = Resistant (Zones size ≦14 mm) M = Moderately sensitive(Zone size 15-19 mm) S = Sensitive (Zone size ≧20 mm)

Intestinal Transit

To determine whether Bifidobacterium longum AH1714 could survive at lowpH values equivalent to those found in the stomach, bacterial cells wereharvested from fresh overnight cultures, washed twice in phosphatebuffer (pH 6.5) and resuspended in TPY broth adjusted to pH 2.5 (with 1MHCl). Cells were incubated at 37° C. and survival measured at intervalsof 5, 30, 60 and 120 minutes using the plate count method. AH1714survived well for 5 minutes at pH 2.5 while no viable cells wererecovered after 30 minutes.

Upon exiting the stomach, putative probiotics are exposed to bile saltsin the small intestine. In order to determine the ability of B. longumAH1714 to survive exposure to bile, cultures were streaked on TPY agarplates supplemented with 0.3% (w/v), 0.5%, 1%, 2%, 5%, 7.5% or 10%porcine bile. B. longum AH1714 growth was observed on plates containingup to 0.5% bile.

TABLE 3 Growth of AH1714 in the presence of porcine bile (duplicateresults) % (w/v) Porcine bile Strain 0.0 0.3 0.5 1.0 2.0 5.0 7.5 10.0 AH1714 +++ ++ + − − − − − +++ = very good growth ~100% ++ = good growth~66% + = poor growth ~33% − = no growth ~0%

In a germ-free murine model, the ability of B. longum AH1714 to transitthe gastrointestinal tract was assessed. Mice consumed 1×10⁹ AH1714daily and faecal pellets were examined for the presence of the fedmicro-organism. Detection of AH1714 was facilitated by isolating aspontaneous rifampicin resistant variant of thebifidobacteria—incorporation of rifampicin in the RCA+cysteine platesused to assess transit ensured that only the fed rifampicin resistantbifiobacteria was cultured. Faecal samples were collected daily and B.longum AH1714 transit through the gastrointestinal tract was confirmed(See FIG. 1)

Anti-Microbial Activity

To assess the antimicrobial activities of B. longum AH1714 againstindicator cultures and to determine if the antimicrobial activity wasdue to acid production, AH1714 was grown overnight in MRS (supplementedwith 0.05% cysteine-HCl). 2 μl of AH1714 culture was spotted onto theagar and incubated for 24 h. The indicator organisms were grown in TSB(E. coli and Salmonella typhimurium), Brucella broth (Campylobacterjejuni) and Reinforced Clostridia Media (RCM, Clostridium difficile).The indicator lawn was prepared by inoculating a molten overlay with 2%(v/v) of the overnight indicator culture, which was poured onto thesurface of the spotted probiotic cultures following overnight growth onthe agar plates. Plates were incubated at 37° C. under suitableconditions for the indicator strain and the growth recorded after 24-48hr. Zones of clearing greater than 1 mm diameter were consideredsensitive to the probiotic strain. This assay was also performed onmedia supplemented with 2% β-glycerophosphate as a buffering agent tolimit antagonistic activity due to acid production.

TABLE 4 antimicrobial activity of AH1714 Zone of inhibition (mm)Indicator strain Non-Buffered Buffered Campylobacter jejuni 9 9Clostridium perfringens 20 10 Salmonella typhimurium 19 11 E. coliO157:H7 16 11

Generation of Rifampicin (Rif^(R)) Resistant Strains of 1714

In order to track transit of AH1714 in faecal samples, a spontaneousrifampicin-resistant variant (rif+) was isolated as follows: a freshbroth culture of AH1714 was spread-plated (100 μL) ontoMRS+rifampicin+cysteine with the lowest concentration of rifampicin(range was 0.1%, 0.08%, 0.06%, 0.04%, 0.02% and 0.002%). Plate mediawithout rifampicin was included as a positive control. Both sets ofplates were incubated anaerobically at 37° C. (48 hours). The removedplates were assessed for purity before picking one colony from therifampicin supplemented agar plate and streaking onto the rifampicinsupplemented plate of next highest concentration. In addition, a colonywas streaked from the MRS agar plate onto a fresh MRS agar plate andboth sets of plates incubated anaerobically at 37° C. (48 hours). Thisprocess was repeated for the full range of rifampicin supplementedplates. A single colony from a fully grown culture on a 50 μg/mLrifampicin supplemented MRS agar plate was used to inoculate into 20 mlMRS broth and the resultant culture used for subsequent stocking. Theidentity of the variant was confirmed by microscopic assessment, IGSsequence analysis and by specific PCR analysis.

Example 2 Congo Red Agar Screen

A Congo red agar screen was used to phenotypically screen for EPSexpressing bacterial strains. Briefly, 10 ml Modified Rogosa broth media(+0.05% cysteine) was inoculated aseptically with a freshly grown colonyof the bacterial strain and incubated anaerobically at 37° C. untilturbid (about 16 to about 24 hours). The broth cultures were asepticallystreaked onto Congo Red Agar plates and incubated anaerobically at 37°C. for 48 hours. It is believed that EPS produced as a by-product of thegrowth and/or metabolism of certain strains prevents the uptake of theCongo red stain resulting in a cream/white colony morphology. Stainsthat produce less EPS take up the Congo red stain easily, resulting in apink/red colony morphology. Strains that do not produce an EPS stain redand look almost transparent in the red agar background.

Referring to FIG. 2 the colony morphology for B. longum AH1714 isconvex, mucoid, bright white colonies

Example 3 Bifidobacteria 1714 Induces a Significantly ElevatedIL-10:IL-12 Ratio

Peripheral blood mononuclear cells (PBMCs) were isolated from healthyhuman peripheral blood using BD Vacutainer CPT tubes (BD catalog362761), as per the manufacturer's instructions. PBMCs were washed andresuspended in Dulbecco's Modified Eagle Medium—Glutamax™ (Glutamax(Glutamine substitute)+pyruvate+4.5 g/l glucose (Gibco catalog10569-010) 10% fetal bovine serum (Sigma catalog F4135), and 1%penicillin/streptomycin (Sigma catalog P0781). PBMCs were incubated(2×10⁵ cells per well) in flat-bottomed 96-well plates and 20 μL of abacterial suspension (at a concentration of 1×10⁷ CFU/mL) was added.PBMCs were co-incubated with bacteria for 48 hours at 37° C./5% CO₂ inan incubator. After the 2 day incubation period, the plates werecentrifuged at 300×g, and the supernatants were removed and storedfrozen at −80° C. until analysis. Interleukin-10 (IL-10) andInterleukin-12p70 (IL-12p70) levels in the culture supernatants werequantified using a 96-well assay kit from Meso Scale Discovery(Gaithersburg, Md.; catalog K15008B-1)

Bacteria were prepared for co-culture experiments in two formats. (a)Freshly grown bacteria were grown in Difco MRS media and harvested justafter entering into stationary phase. All cells were grown underanaerobic conditions at 37° C. (b) Bacteria were grown under anaerobicconditions at 37° C. in Difco MRS media and harvested just afterentering into stationary phase. Freeze dried powders were generated foreach of these bacteria and stored at −80° C. in pre-aliquoted 100 mgvials. Immediately prior to their use, one aliquot of each strain wasremoved from the freezer and allowed to reach room temperature. Eachstrain was washed 3 times in 10 ml ringers followed by centrifugation. Afresh vial was used on each occasion. Growth curves (OD vs number oflive cells) were constructed for each growth condition, and washed cellswere normalized by cell number before addition to the PBMCs. Ano-bacteria control was also included in all experiments. All assayswere done in triplicate. The results are presented in FIG. 3. Thecontrol of inflammatory diseases is exerted at a number of levels. Thecontrolling factors include hormones, prostaglandins, reactive oxygenand nitrogen intermediates, leukotrienes and cytokines. Cytokines arelow molecular weight biologically active proteins that are involved inthe generation and control of immunological and inflammatory responses.A number of cell types produce these cytokines, with neutrophils,monocytes and lymphocytes being the major sources during inflammatoryreactions due to their large numbers at the injured site. Multiplemechanisms exist by which cytokines generated at inflammatory sitesinfluence the inflammatory response. Chemotaxis stimulates homing ofinflammatory cells to the injured site, whilst certain cytokines promoteinfiltration of cells into tissue. Cytokines released within the injuredtissue result in activation of the inflammatory infiltrate. Mostcytokines are pleiotropic and express multiple biologically overlappingactivities. As uncontrolled inflammatory responses can result indiseases such as IBD, it is reasonable to expect that cytokineproduction has gone astray in individuals affected with these diseases.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine which isproduced by many cell types including monocytes, macrophages, dendriticcells, mast cells and lymphocytes (in particular T regulatory cells).IL-10 down-regulates the expression of pro-inflammatory Th1 cytokines,MHC class II antigens, and co-stimulatory molecules on antigenpresenting cells. It also enhances B cell survival, proliferation, andantibody production. This cytokine can block NF-κB activity, and isinvolved in the regulation of the JAK-STAT signaling pathway. Murineknock-out studies have demonstrated the essential role for IL-10 inimmunoregulation as IL-10KO mice develop severe colitis. In addition,bacteria which are potent inducers of IL-10 have been shown to promote Tregulatory cell differentiation in vivo thus contributing toimmunological homeostasis (7; 8). Interleukin-12 (IL-12) is apro-inflammatory cytokine associated with polarisation of Th1 effector Tcell responses and stimulates the production of other pro-inflammatoryTh1 cytokines, such as interferon-gamma (IFN-γ) and tumor necrosisfactor-alpha (TNF-α), from T and natural killer (NK) cells. High levelsof IL-12 expression is associated with autoimmunity. Administration ofIL-12 to people suffering from autoimmune diseases was shown to worsendisease symptoms. In contrast, IL-12 knock-out mice or treatment of micewith IL-12 neutralising antibodies ameliorated the disease.

Cytokine cascades and networks control the inflammatory response, ratherthan the action of a particular cytokine on a particular cell type. Therelative levels of expression, or balance, of two cytokines (such asIL-10 and IL-12) is more informative than the expression of a singlecytokine. In these studies, we stimulated human PBMCs with a range ofdifferent bacterial strains. All strains induced IL-10 and all strainsinduced IL-12. However, examination of the ratio between IL-10 and IL-12induction revealed that some bacterial strains induced a higher ratio(i.e. more IL-10 with less IL-12) compared to other strains. This is ameaningful observation as it is the balance between each of theseopposing signals that ultimately determines the immunological outcome.It is anticipated that a high IL-10:IL-12 ratio would promote ananti-inflammatory response associated with appropriate immunoregulatoryactivity while a low IL-10:IL-12 ratio would contribute to Th1polarisation of the immune response. Thus, the PBMC IL-10:IL-12 ratio isa important selection criterion for identification of bacterial strainswith immunoregulatory properties.

Example 4 Long Term Feeding of Mice with Bif

AH1714 is associated with increased anti-inflammatory cytokine IL-10 andwith decreased pro-inflammatory and Th1 cytokines TNF-α□ IFN-γ and IL-12in healthy animals and in a model of Sepsis/Inflammation.

Materials & Methods:

Female Balb/c mice@6-8 weeks of age are sourced from Harlan UK andhoused in individually ventilated cages and provided ad libitum accessto sterile standard mouse chow and water.

Mice of similar weight are randomised into 2 groups and administered PBS(carrier control n=9), Bifidobacterium longum strain AH1714 (n=17) viaoral gavage on a daily basis for 115 days. Following the period ofadministration blood is sampled from 10 AH1714 mice and 6 carriercontrols were challenged with 1 mg/kg LPS (Sigma, L4391) via intraperitoneal injection.

Following the period of administration blood is sampled from 6 AH1714mice and 4 carrier controls, serum is extracted and preserved forcytokine measurements. Spleens are also removed and single cellsuspensions cultured in vitro. Cytokines are measured in cellsupernatants following 48 hours culturing.

A further 10 AH1714 mice and 6 control mice are administered a singledose of LPS@1 mg/kg via intraperitoneal injection. After 2 hours bloodis sampled and the mice were culled. Serum and splenocyte cells weretreated and analysed as previously described.

Splenocyte Cytokine Assay

Splenocytes are isolated from spleens and incubated for 48 hours at 37°C. (in the presence of penicillin and streptomycin) with control media,LPS, or antiCD3/CD28. Cytokines in the culture supernatants are assayedusing a 96-well assay kit from Meso Scale Discovery (Gaithersburg, Md.;catalog K15008B-1). Interleukin 1 beta (Il-1b), Interleukin 6 (Il-6),Interleukin 8 (Il-8) Interleukin 10 (Il-10), Interleukin 12p70(Il12p70), Interferon-gamma (IFN-γ) and Tumor Necrosis Factor alpha(TNF□) are quantitated and reported as picograms per millilitre (pg/mL).

Serum Cytokine Assay

Serum is analysed using the Meso Scale Discovery mouse IL-10 and TNF-αUltrasensitive kit.

Results

Long term feeding of mice (115 days) with Bif. AH1714 is associated withan increase in the anti-inflammatory cytokine IL-10 from stimulated exvivo PBMCs, compared to placebo group (fed PBS) for healthy mice (SeeFIG. 4 (B)) or in a Sepsis/Inflammation model (mice challenged with LPS;See FIG. 4 (C)).

Long term feeding of mice (115 days) with Bif. AH1714 is associated witha decrease in the pro-inflammatory and Th1 cytokines TNF-α□ IFN-γ andIL-12 (p70 sub unit) from stimulated ex vivo PBMCs, compared to placebogroup (fed PBS) in a Sepsis/Inflammation model (mice challenged withLPS; See FIG. 5 (B), FIG. 6 (B) and FIG. 7 (B)).

Long term feeding mice (115 days) with Bif. AH1714 is associated with anincrease in the serum levels of anti-inflammatory cytokine IL-10 and adecrease in the pro-inflammatory and Th1 cytokine TNF-α, compared toplacebo group (fed PBS) in a Sepsis/Inflammation model (mice challengedwith LPS; See FIG. 8 (A & B)).

Taken together, these results demonstrate that Bifidobacterium longumstrain 1714 has in-vivo systemic immunomodulatory and anti-inflammatoryactivity and protects against LPS or TLR-4 mediated inflammatoryresponses.

Example 5 Bif 1714 has Immunomodulatory Activity when Co-Incubated withHuman Immune System Cells In Vitro, Different to that of Bif. AH35624.Materials & Methods

Bifidobacterium longum infantis strain UCC35624 (B624), two independentculture batches (1 & 2) and Bifidobacterium longum strain 1714 isassayed using a PBMC cytokine induction assay. Bacteria are prepared forco-culture experiments in the following formats. Bacteria are grownunder anaerobic conditions at 37° C. in Difco MRS Media and harvestedjust after entering into stationary phase. Freeze dried powders aregenerated for each of these bacteria and stored at −80° C. inpre-aliquoted 100 mg vials. Immediately prior to their use, one aliquotof each strain is removed from the freezer and allowed to reach roomtemperature. Each strain is washed 3 times in 10 ml ringers followed bycentrifugation. A fresh vial is used on each occasion.

Direct microscopic counts are performed using a Petroff-Hausser countingchamber as per the manufacturer's instructions and washed cellsnormalized by cell number before addition to the PBMC assay. Bacteria(20 μl in phosphate buffered saline (PBS)) are added to each well ofPBMCs to give the total number of bacteria as indicated for eachexperiment.

PBMC (Peripheral Blood Mononuclear Cell) Cytokine Induction Assay

Peripheral blood mononuclear cells (PBMCs) are isolated from healthyhuman peripheral blood using BD Vacutainer CPT tubes (BD catalog362761), as per the manufacturer's instructions. PBMCs are washed andresuspended in Dulbecco's Modified Eagle Medium—Glutamax™ (Glutamax(Glutamine substitute)+pyruvate+4.5 g/l glucose (Gibco catalog10569-010) 10% fetal bovine serum (Sigma catalog F4135), and 1%penicillin/streptomycin (Sigma catalog P0781). PBMCs are incubated(2×10⁵ cells per well) in flat-bottomed 96-well plates and 20 μL of abacterial suspension. A no-bacteria control also is run. All assays aredone in triplicate. After a 2-day incubation at 37° C., the plates werespun at 300×g, and the supernatants were removed and stored frozen at−80° C. until analysis. PBMCs are co-incubated with bacteria for 48hours at 37° C./5% CO² in an incubator. After the 2 day incubationperiod, the plates are centrifuged at 300×g, and the supernatantsremoved and stored frozen at −80° C. until analysis. Cytokines in theculture supernatants are assayed using a 96-well assay kit from MesoScale Discovery (Gaithersburg, Md.; catalog K15008B-1). HumanInterleukin 1 beta (Il-1b), Human Interleukin 6 (Il-6), HumanInterleukin 8 (Il-8) Human Interleukin 10 (Il-10), Human Interleukin12p70 (Il12p70), Human Interferon-gamma (IFN-γ) and Human Tumor NecrosisFactor alpha (TNFα) are quantitated and reported as picograms permillilitre (pg/mL). Each sample is assayed in duplicate.

Results

Bifidobacterium longum infantis strain UCC35624 (B624), two independentculture batches (1 & 2) and Bifidobacterium longum strain 1714 areassayed for immuno-modulation using a PBMC cytokine induction assay with1.0E+07 bacteria. Supernatants are assayed for a range of cytokines,including IL-1β, -6, -8, -10 and -12, TNF-α□ and IFN-γ.

By comparison with 35624 (both cultures of which gave a similar patternfor all cytokines measured), strain 1714 exhibited a very similarpattern for many of the cytokines measured. Surprisingly however, 1714gave quite a different pattern for IL-12, IFNγ and IL-6.

IL-6: Incubation with 1714 induces a significantly lower level of IL-6compared to 35624 at 1.0×10⁷ bacteria per well (See Table 5)

TABLE 5 Strain (1 × 10E7 bacteria) IL-6 (~pg/ml) 35624 28,000 171416,000

IL-12: Incubation with 1714 induces a significantly lower level of IL-12compared to 35624 at 1.0×10⁷ bacteria per well (See Table 6)

INF-γ Incubation with 1714 induces a significantly lower level of INF-γcompared to 35624 at 1.0×10⁷ bacteria per well (See Table 6)

TABLE 6 Strain (1 × 10E7 IL-12 IL-12 IFN-α IFN-γ bacteria) (~pg/ml)(~pg/ml) (~pg/ml) (~pg/ml) 35624 500 180 5000 1500 1714 (1) 220 115 2400750 1714 (2) 240 80 2600 400

Foligne et al¹⁹, have demonstrated that lactic acid bacteria strainsdisplaying an in vitro capacity to induce higher levels of theanti-inflammatory cytokine IL-10 and lower levels of the inflammatorycytokine IL-12 offered the best protection in the in vivo colitis modelwhereas in contrast, strains leading to a low IL-10/IL-12 cytokine ratiocould not significantly alleviate colitis symptoms. The in vivoprotection observed was strain specific. The cytokine profile obtainedfor Bif. AH1714 would suggest that this strain has the potential forimproved efficacy in the in vivo ulcerative colitis model.

IL-6 is a cytokine strongly implicated in the pathology of IBS. L-6 isrelevant to many disease processes such as diabetes, atherosclerosis,depression, Alzheimer's Disease, systemic lupus erythematosus andrheumatoid arthritis. Hence there is an interest in developing anti-IL-6agents as therapy against many of these diseases.

Example 6 Bif. AH1714 Reduces LPS-Induced NFκB Activity in an In-VivoMurine Sepsis/Inflammation Model Materials & Methods

NFkBlux transgenic mice on a C57BL/6J-CBA/J background are obtained fromCharles River Laboratories (Wilmington, USA) and bred in-house. Mice arehoused under barrier maintained conditions.

Female animals are administered Bif. AH1714, as a freeze-dried powderreconstituted in water at approximately 1×10⁹ colony formingunits/day/animal, or a placebo control. Mice consume the commensalmicro-organism in their drinking water ad libitum for 20 days prior toLPS challenge.

NFkB activity is measured following the administration of the substrateluciferin and imaged using the Xenogen IVIS 100. Baseline NFkB activityis measured prior to challenge with a single 0.5 mg/kg dose of LPS.After 3 hours all animals are then reimaged. Whole body NFkB activity isassessed by subtracting baseline readings.

All animals are then culled and spleens, livers, small intestine andcolon removed and placed in a culture dish for individual imaging.

Results

Bif. AH1714 reduces systemic LPS-induced NFkB activity in an in-vivomurine Sepsis/Inflammation model as demonstrated by a decreased NFkBactivity in spleens isolated 3 hours post challenge (See FIG. 9) andfrom whole animal imaging 1.5 hours post challenge (See FIG. 10) from1714-fed animals compared to Placebo-fed animals. These resultsdemonstrate that feeding with Bif. 1714 is associated with a decreasedlevel of systemic inflammation associated with the transcription factorNFkB.

Example 7 1714 Exhibits Positive Benefits in Animal Models of Depressionand Anxiety

Depression and anxiety are the most common psychiatric disorders with ahigh prevalence rate in the community. Anxiety disorders are usuallysubdivided into panic disorder, generalised anxiety disorder,post-traumatic disorder and obsessive compulsive disorder.

Modern antidepressants such as the selective serotonin reuptakeinhibitors (e.g. fluoxetine) and selective noradrenergic andserotinergic reuptake inhibitors (e.g. venlafaxine) are widely used totreat these disorders. However, the treatments are not always effectiveand are not acceptable to patients. There is a need to developalternative strategies. The possibility that probiotics might beeffective in such conditions is suggested by previous data indicatingthat the probiotic Bifidobacterium Infantis reduces the stress hormonecorticosterone in rodents (9).

We here examine the behavioural effects of Bifidobacterium AH1714 inmodels of stress in mice and compared with a widely used SSRI, namely,escitalopram, which is used to treat both anxiety and depression.Animals are treated with either escitalopram or AH1714 for three weeks.

Material & Methods Tail Suspension Test

A well characterized test for assessing depression-like andantidepressant like activity. Mice are individually suspended by thetail to a horizontal ring-stand bar (distance from floor=30 cm) usingadhesive tape (distance from tip of tail=2 cm). Typically, micedemonstrate several escape-oriented behaviours interspersed withtemporally increasing bouts of immobility. A 6-minute test session isemployed which is videotaped. Videotapes are subsequently scored by ahighly trained observer who is unaware of the treatment. The parameterrecorded is the number of seconds spent immobile.

Fear Conditioning Test

Widely used to assess the cognitive components of anxiety disorders. Weuse a three day protocol which allows for contextual and cue-associatedfear learning to be observed. Following 3 minutes of exploring theirenvironmental context mice receiving 6 pairings of 20 seconds of aspecific cue (Tone of 10 KHz, 70 dB combined with apparatus light on)coupled at the end with 2 seconds of a mild electrical footshock (0.4mA), this is followed by 1 min exposure to the context only. Theprocedure is repeated for two subsequent days, however, no shock isgiven and freezing behaviour to the context or cue is observedthroughout. The first day allows for assessment of the ability of theintervention to alter the strength of context and cue-induced fearconditioning, whereas the third day allows for extinction of fearlearning to be observed. Extinction is the formation of new memories anddrugs that facilitate extinction may play a role in the treatment ofpost-traumatic stress disorder.

The Marble-Burping Test

Proposed as model of obsessive compulsive disorder. Animals who are moreanxious must engage in active behaviours (defensive marble burying) toavoid anxiogenic stimuli in the light-dark box and elevated mazes. Miceare placed individually in small cages, in which 20 marbles had beenequally distributed on top of a 5 cm-deep bed of sawdust, and a wire lidplaced on top of the cage. Mice are left undisturbed for 30 min, afterwhich the number of buried marbles (i.e., those more than less thanthree-quarters covered by sawdust) are counted.

Results

In the tail suspension test AH1714 gives a positive result suggestingpossible antidepressant activity. Referring to FIG. 11, AH1714 inducedlower immobility time than the vehicle (Veh) which suggests lowerdepression-like behavious in 1714-fed animals. This is similar to theimpact of conventional antidepressants such as Lexapro®.

In terms of cognition, in the fear conditioning test, test animalstreated with AH1714 showed a positive learning effect. Referring to FIG.12, in context (mainly hippocampus and amygdale-dependent memories) fearconditioning tests, 1714 induced higher freezing to the context (Cxt)than the vehicle (Veh) on day 1 and day 2, with the same freezingpercentage as the vehicle on day 3, this suggests that 1714 promotescontextual fear learning and memory without impairing extinction,suggesting a positive role in contextual memory of fearful events.Referring to FIG. 13, in cue (amygdale-dependent) fear conditioningtests, 1714 induced higher freezing to the fearful cue (stimulus) thanthe vehicle (Veh) on day 1, with the same freezing percentage as thevehicle on days 2 and 3. This suggests that 1714 promotedamygdale-dependent (cue) fear learning and memory without impairingmemory and extinction, suggesting a positive role in the memory of afearful stimulus independently of the context.

Evidence of a possible effect in obsessive compulsive disorder emergesfrom studies with the marble burying test. Animals treated with AH1714buried less marbles in the marble burying task which is indicative oflower anxiety in 1714-fed animals and suggests a possible effect inobsessive compulsive disorder (FIG. 14). As in the case of escitalopram,AH1714 induced a lower body temperature increase induced by the stressof being handled (decreased stress induced hypothermia) this suggestslower anxiety in 1714-fed animals (FIG. 24). There were no differencesbetween the results with either intervention.

Conclusion

AH1714 in animal models of depression and anxiety behaves in a similarway to a conventional antidepressant. The impact observed is similar tothat reported in the literature for antidepressants such as SSRIs.

Overall, the data indicate that AH1714 may be of benefit in thetreatment of the psychiatric syndromes of depression and anxiety.

Example 8 1714 Exhibits Positive Benefits on Inflammatory Markers inDiet-Induced Obesity

In recent years, it has been well established that obesity is associatedwith a low-grade inflammation that contributes to the development of thepathologies associated with obesity which include type 2 diabetesmellitus (T2D), cardiovascular disease (CVD), hypertension,hypercholesterolemia, hypertriglyceridemia, and non-alcoholic fattyliver disease (NAFLD). Visceral fat produces a number of inflammatorycytokines and chemokines (such as leptin, tumor necrosis factor-α(TNF-α), macrophage chemo-attractant protein-1 and interleukin-6, amongothers), whose production can be pathologically dysregulated in theobese state (reviewed by Shoelson et al., 2007). Indeed, whilemacrophages are thought to contribute in an important manner to insulinresistance, other studies have suggested that harnessing theanti-inflammatory properties of cells with a potentially regulatoryphenotype may have therapeutic potential. A recent study suggests thatT_(reg) cells reduce the inflammatory state of adipose tissue and, thus,insulin resistance in mice (Feurer et al., 2009). In addition, a seminalbody of work has implicated abnormalities of the gut microbiota as adriving force of obesity-related metabolic dysregulation, suggestingthat interventions which target gut health will have beneficial healtheffects in obesity related metabolic derangements. It has been suggestedthat the gut microbiota may be involved in the development of obesity inthe regulation of energy homeostasis, in insulin resistance,non-alcoholic fatty liver disease and in energy, lipid and amino acidmetabolism (reviewed by Ley et al., 2009)

The Diet-Induced Obesity (DIO) mouse model was chosen as the mostappropriate mouse model for assessing the impact of selected probioticcandidates on obesity and metabolic health and to look at therelationship between obesity and inflammatory markers. The DIO mousemodel refers to healthy mice fed a high-fat diet to induce obesity overtime.

Experimental Design

Seven-week old male C57BL/J6 mice were fed a low-fat diet (10% caloriesfrom fat; Research Diets, New Jersey; #D12450B), a high-fat diet (DIO;45% calories from fat; Research Diets, New Jersey; #D12451) or ahigh-fat diet with AH1714 (1×10⁹ cfu/day) in drinking water for 14weeks. All mice were housed in groups of 5 and fresh probiotic aliquotswere administered daily. Body weight and food intake were assessedweekly. At the end of 14 weeks the mice were sacrificed and internalorgans were removed, weighed and stored at −80° C. The spleens removedand splenocyte cytokine assays were carried out as in example 4.

Results

As expected, DIO mice gained significantly more fat mass (p<0.001)compared to lean controls over the 14-week feeding period. In agreementwith previous studies, DIO mice consumed significantly more caloriesthan lean controls, as measured by the cumulative caloric intake overthe 14 week period of the study (p<0.001). In LPS stimulated splenocytes(innate immunity stimulus) from DIO mice, AH1714 had the effect oflowering the TNFα and IL-12 cytokine response to LPS (FIG. 16). InCD3/CD28 stimulated spenocytes (adaptive immunity stimulus), treatmentwith AH1714 had the effect of lowering the IL6 cytokine response. Theseresults indicate a systemic anti-inflammatory effect in the DIO mousemodel consistent with the PBMC data and in vivo mouse model dataillustrated in other examples.

Immunomodulation

The human immune system plays a significant role in the aetiology andpathology of a vast range of human diseases. Hyper and hypo-immuneresponsiveness results in, or is a component of, the majority of diseasestates. One family of biological entities, termed cytokines, areparticularly important to the control of immune processes. Pertubancesof these delicate cytokine networks are being increasingly associatedwith many diseases. These diseases include but are not limited toinflammatory disorders, immunodeficiency, inflammatory bowel disease,irritable bowel syndrome, cancer (particularly those of thegastrointestinal and immune systems), diarrhoeal disease, antibioticassociated diarrhoea, paediatric diarrhoea, appendicitis, autoimmunedisorders, multiple sclerosis, Alzheimer's disease, rheumatoidarthritis, coeliac disease, diabetes mellitus, organ transplantation,bacterial infections, viral infections, fungal infections, periodontaldisease, urogenital disease, sexually transmitted disease, HIVinfection, HIV replication, HIV associated diarrhoea, surgicalassociated trauma, surgical-induced metastatic disease, sepsis, weightloss, anorexia, fever control, cachexia, wound healing, ulcers, gutbarrier function, allergy, asthma, respiratory disorders, circulatorydisorders, coronary heart disease, anaemia, disorders of the bloodcoagulation system, renal disease, disorders of the central nervoussystem, hepatic disease, ischaemia, nutritional disorders, osteoporosis,endocrine disorders, epidermal disorders, psoriasis and acne vulgaris.

The effects on cytokine production are specific for each of theprobiotic strains examined. Thus specific probiotic strains may beselected for normalising an exclusive cytokine imbalance particular fora specific disease type. Customisation of disease specific therapies canbe accomplished using either a single strain of AH1714 or mutants orvariants thereof or a selection of these strains.

Immune Education

The enteric flora is important to the development and proper function ofthe intestinal immune system. In the absence of an enteric flora, theintestinal immune system is underdeveloped, as demonstrated in germ freeanimal models, and certain functional parameters are diminished, such asmacrophage phagocytic ability and immunoglobulin production (10). Theimportance of the gut flora in stimulating non-damaging immune responsesis becoming more evident. The increase in incidence and severity ofallergies in the western world has been linked with an increase inhygiene and sanitation, concomitant with a decrease in the number andrange of infectious challenges encountered by the host. This lack ofimmune stimulation may allow the host to react to non-pathogenic, butantigenic, agents resulting in allergy or autoimmunity. Deliberateconsumption of a series of non-pathogenic immunomodulatory bacteriawould provide the host with the necessary and appropriate educationalstimuli for proper development and control of immune function.

Inflammation

Inflammation is the term used to describe the local accumulation offluid, plasma proteins and white blood cells at a site that hassustained physical damage, infection or where there is an ongoing immuneresponse. Control of the inflammatory response is exerted on a number oflevels (11). The controlling factors include cytokines, hormones (e.g.hydrocortisone), prostaglandins, reactive intermediates andleukotrienes. Cytokines are low molecular weight biologically activeproteins that are involved in the generation and control ofimmunological and inflammatory responses, while also regulatingdevelopment, tissue repair and haematopoiesis. They provide a means ofcommunication between leukocytes themselves and also with other celltypes. Most cytokines are pleiotrophic and express multiple biologicallyoverlapping activities. Cytokine cascades and networks control theinflammatory response rather than the action of a particular cytokine ona particular cell type (12). Waning of the inflammatory response resultsin lower concentrations of the appropriate activating signals and otherinflammatory mediators leading to the cessation of the inflammatoryresponse. TNFα is a pivotal proinflammatory cytokine as it initiates acascade of cytokines and biological effects resulting in theinflammatory state. Therefore, agents which inhibit TNFα are currentlybeing used for the treatment of inflammatory diseases, e.g. infliximab.

Pro-inflammatory cytokines are thought to play a major role in thepathogenesis of many inflammatory diseases, including inflammatory boweldisease (IBD). Current therapies for treating IBD are aimed at reducingthe levels of these pro-inflammatory cytokines, including IL-8 and TNFα.Such therapies may also play a significant role in the treatment ofsystemic inflammatory diseases such as rheumatoid arthritis.

The strains of the present invention may have potential application inthe treatment of a range of inflammatory diseases, particularly if usedin combination with other anti-inflammatory therapies, such asnon-steroid anti-inflammatory drugs (NSAIDs) or Infliximab.

Cytokines and Cancer

The production of multifunctional cytokines across a wide spectrum oftumour types suggests that significant inflammatory responses areongoing in patients with cancer. It is currently unclear what protectiveeffect this response has against the growth and development of tumourcells in vivo. However, these inflammatory responses could adverselyaffect the tumour-bearing host. Complex cytokine interactions areinvolved in the regulation of cytokine production and cell proliferationwithin tumour and normal tissues (13, 14). It has long been recognizedthat weight loss (cachexia) is the single most common cause of death inpatients with cancer and initial malnutrition indicates a poorprognosis. For a tumour to grow and spread it must induce the formationof new blood vessels and degrade the extracellular matrix. Theinflammatory response may have significant roles to play in the abovemechanisms, thus contributing to the decline of the host and progressionof the tumour. Due to the anti-inflammatory properties ofBifidobacterium longum infantis these bacterial strains they may reducethe rate of malignant cell transformation. Furthermore, intestinalbacteria can produce, from dietary compounds, substances with genotoxic,carcinogenic and tumour-promoting activity and gut bacteria can activatepro-carcinogens to DNA reactive agents (15). In general, species ofBifidobacterium have low activities of xenobiotic metabolizing enzymescompared to other populations within the gut such as bacteroides,eubacteria and clostridia. Therefore, increasing the number ofBifidobacterium bacteria in the gut could beneficially modify the levelsof these enzymes.

Vaccine/Drug Delivery

The majority of pathogenic organisms gain entry via mucosal surfaces.Efficient vaccination of these sites protects against invasion by aparticular infectious agent. Oral vaccination strategies haveconcentrated, to date, on the use of attenuated live pathogenicorganisms or purified encapsulated antigens (16). Probiotic bacteria,engineered to produce antigens from an infectious agent, in vivo, mayprovide an attractive alternative as these bacteria are considered to besafe for human consumption (GRAS status).

Murine studies have demonstrated that consumption of probiotic bacteriaexpressing foreign antigens can elicit protective immune responses. Thegene encoding tetanus toxin fragment C (TTFC) was expressed inLactococcus lactis and mice were immunized via the oral route. Thissystem was able to induce antibody titers significantly high enough toprotect the mice from lethal toxin challenge. In addition to antigenpresentation, live bacterial vectors can produce bioactive compounds,such as immunostimulatory cytokines, in vivo. L. lactis secretingbioactive human IL-2 or IL-6 and TTFC induced 10-15 fold higher serumIgG titres in mice immunized intranasally (17). However, with thisparticular bacterial strain, the total IgA level was not increased bycoexpression with these cytokines. Other bacterial strains, such asStreptococcus gordonii, are also being examined for their usefulness asmucosal vaccines. Recombinant S. gordonii colonizing the murine oral andvaginal cavities induced both mucosal and systemic antibody responses toantigens expressed by this bacterial (18). Thus oral immunization usingprobiotic bacteria as vectors would not only protect the host frominfection, but may replace the immunological stimuli that the pathogenwould normally elicit thus contributing to the immunological educationof the host.

Prebiotics

The introduction of probiotic organisms is accomplished by the ingestionof the micro-organism in a suitable carrier. It would be advantageous toprovide a medium that would promote the growth of these probioticstrains in the large bowel. The addition of one or moreoligosaccharides, polysaccharides, or other prebiotics enhances thegrowth of lactic acid bacteria in the gastrointestinal tract. Prebioticsrefers to any non-viable food component that is specifically fermentedin the colon by indigenous bacteria thought to be of positive value,e.g. bifidobacteria, lactobacilli. Types of prebiotics may include thosethat contain fructose, xylose, soya, galactose, glucose and mannose. Thecombined administration of a probiotic strain with one or more prebioticcompounds may enhance the growth of the administered probiotic in vivoresulting in a more pronounced health benefit, and is termed synbiotic.

Other Active Ingredients

It will be appreciated that the probiotic strains may be administeredprophylactically or as a method of treatment either on its own or withother probiotic and/or prebiotic materials as described above. Inaddition, the bacteria may be used as part of a prophylactic ortreatment regime using other active materials such as those used fortreating inflammation or other disorders especially those with animmunological involvement. Such combinations may be administered in asingle formulation or as separate formulations administered at the sameor different times and using the same or different routes ofadministration.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

REFERENCES

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1. An isolated strain of Bifidobacterium NCIMB
 41676. 2. ABifidobacterium strain as claimed in claim 1 in the form of viablecells.
 3. A Bifidobacterium strain as claimed in claim 1 in the form ofnon-viable cells.
 4. A Bifidobacterium strain as claimed in any ofclaims 1 to 3 wherein the Bifidobacterium is isolated from colonicbiopsy tissue from a healthy human subject.
 5. A Bifidobacterium strainas claimed in any of claims 1 to 4 wherein the strain is significantlyimmunomodulatory following oral consumption in humans.
 6. A formulationwhich comprises a Bifidobacterium strain as claimed in any of claims 1to
 5. 7. A formulation as claimed in claim 6 which further comprises aprobiotic material.
 8. A formulation as claimed in claim 6 or 7 whichfurther comprises a prebiotic material.
 9. A formulation as claimed inany one of claims 6 to 8 further comprising an ingestable carrier.
 10. Aformulation as claimed in claim 9 wherein the ingestable carrier is apharmaceutically acceptable carrier such as a capsule, tablet or powder.11. A formulation as claimed in claim 9 wherein the ingestable carrieris a food product such as acidified milk, yoghurt, frozen yoghurt, milkpowder, milk concentrate, cheese spreads, dressings or beverages.
 12. Aformulation as claimed in any one of claims 6 to 11 which furthercomprises a protein and/or peptide, in particular proteins and/orpeptides that are rich in glutamine/glutamate, a lipid, a carbohydrate,a vitamin, mineral and/or trace element.
 13. A formulation as claimed inany one of claims 6 to 12 wherein the Bifidobacterium strain is presentin an amount of more than 10⁶ cfu per gram of the formulation.
 14. Aformulation as claimed in any one of claims 6 to 13 which furthercomprises an adjuvant.
 15. A formulation as claimed in any one of claims6 to 14 which further comprises a bacterial component.
 16. A formulationas claimed in any one of claims 6 to 15 which further comprises a drugentity.
 17. A formulation as claimed in any one of claims 6 to 16 whichfurther comprises a biological compound.
 18. A formulation as claimed inany one of claims 6 to 17 for immunisation and vaccination protocols.19. A Bifidobacterium strain as claimed in any one of claims 1 to 5 or aformulation as claimed in any one of claims 6 to 18 for use infoodstuffs.
 20. A Bifidobacterium strain as claimed in any one of claims1 to 5 or a formulation as claimed in any one of claims 6 to 18 for useas a medicament.
 21. A Bifidobacterium strain as claimed in any one ofclaims 1 to 5 or a formulation as claimed in any one of claims 6 to 18for use in the prophylaxis and/or treatment of undesirable inflammatoryactivity.
 22. A Bifidobacterium strain as claimed in any one of claims 1to 5 or a formulation as claimed in any of claims 6 to 18 for use in theprophylaxis and/or treatment of undesirable gastrointestinalinflammatory activity such as inflammatory bowel disease eg. Crohnsdisease or ulcerative colitis, irritable bowel syndrome; pouchitis; orpost infection colitis.
 23. A Bifidobacterium strain as claimed in anyone of claims 1 to 5 or a formulation as claimed in any one of claims 6to 18 for use in the prophylaxis and/or treatment of gastrointestinalcancer(s).
 24. A Bifidobacterium strain as claimed in any one of claims1 to 5 or a formulation as claimed in any one of claims 6 to 18 for usein the prophylaxis and/or treatment of systemic disease such asrheumatoid arthritis.
 25. A Bifidobacterium strain as claimed in any oneof claims 1 to 5 or a formulation as claimed in any one of claims 6 to18 for use in the prophylaxis and/or treatment of autoimmune disordersdue to undesirable inflammatory activity.
 26. A Bifidobacterium strainas claimed in any one of claims 1 to 5 or a formulation as claimed inany one of claims 6 to 18 for use in the prophylaxis and/or treatment ofcancer due to undesirable inflammatory activity.
 27. A Bifidobacteriumstrain as claimed in any one of claims 1 to 5 or a formulation asclaimed in any one of claims 6 to 18 for use in the prophylaxis ofcancer.
 28. A Bifidobacterium strain as claimed in any one of claims 1to 5 or a formulation as claimed in any one of claims 6 to 18 for use inthe prophylaxis and/or treatment of diarrhoeal disease due toundesirable inflammatory activity, such as Clostridium difficileassociated diarrhoea, Rotavirus associated diarrhoea or post infectivediarrhoea or diarrhoeal disease due to an infectious agent, such as E.coli.
 29. A Bifidobacterium strain as claimed in any one of claims 1 to5 or a formulation as claimed in any one of claims 6 to 18 for use inthe preparation of anti-inflammatory biotherapeutic agents for theprophylaxis and/or treatment of undesirable inflammatory activity. 30.Bifidobacterium strains as claimed in claim 29 for use in thepreparation of a panel of biotherapeutic agents for modifying the levelsof IL-10.
 31. A Bifidobacterium strain as claimed in any one of claims 1to 5 or a formulation as claimed in any one of claims 6 to 18 for use inthe prevention and/or treatment of inflammatory disorders,immunodeficiency, inflammatory bowel disease, irritable bowel syndrome,cancer (particularly of the gastrointestinal and immune systems),diarrhoeal disease, antibiotic associated diarrhoea, paediatricdiarrhoea, appendicitis, autoimmune disorders, multiple sclerosis,Alzheimer's disease, rheumatoid arthritis, coeliac disease, diabetesmellitus, organ transplantation, bacterial infections, viral infections,fungal infections, periodontal disease, urogenital disease, sexuallytransmitted disease, HIV infection, HIV replication, HIV associateddiarrhoea, surgical associated trauma, surgical-induced metastaticdisease, sepsis, weight loss, anorexia, fever control, cachexia, woundhealing, ulcers, gut barrier function, allergy, asthma, respiratorydisorders, circulatory disorders, coronary heart disease, anaemia,disorders of the blood coagulation system, renal disease, disorders ofthe central nervous system, hepatic disease, ischaemia, nutritionaldisorders, osteoporosis, endocrine disorders, epidermal disorders,psoriasis, acne vulgaris, panic disorder, behavioral disorder and/orpost traumatic stress disorders.
 32. A Bifidobacterium strain as claimedin any one of claims 1 to 5 wherein the strain acts by antagonising andexcluding proinflammatory micro-organisms from the gastrointestinaltract.
 33. A Bifidobacterium strain as claimed in any one of claims 1 to5 or a formulation as claimed in any one of claims 6 to 18 for use inthe preparation of anti-inflammatory biotherapeutic agents for reducingthe levels of pro inflammatory cytokines.
 34. Use of a Bifidobacteriumstrain as claimed in any one of claims 1 to 5 as an anti-infectiveprobiotic strain.
 35. A Bifidobacterium strain as claimed in any one ofclaims 1 to 5 or a formulation as claimed in any one of claims 6 to 18for use in the prophylaxis and/or treatment of bipolar illness,depression, mood disorders, and/or anxiety disorders.
 36. Use of aBifidobacterium strain as claimed in any one of claims 1 to 5 or aformulation as claimed in any one of claims 6 to 18 as a cognativeenhancer for the prophylaxis and/or treatment of disorders of thecentral nervous system such as Alzheimer's disease, schizophrenia and/ormild cognative disorder.
 37. Use of a Bifidobacterium strain as claimedin any one of claims 1 to 5 or a formulation as claimed in any one ofclaims 6 to 18 for the prophylaxis and/or treatment of obesity relatedinflammation.
 38. Use of a Bifidobacterium strain as claimed in any oneof claims 1 to 5 or a formulation as claimed in any one of claims 6 to18 for the prophylaxis and/or treatment of obesity related metabolicdysregulation.